Composition and method for treating diseased nails

ABSTRACT

A nail-permeable medication means is disclosed for delivering a medicament through nails, claws, hoofs, or other similar hardened tissue of dermal derivation. The nail-permeable medication means includes a proteolytic enzyme component which facilitates permeation of substances through the hardened nail or keratin tissue, and also includes a medicament component selected to treat a specific disease. The nail-permeable medication means is particularly useful for treating onychomycosis of the fingernail or toenail, and avoids the need for more drastic therapeutic modalities, such as removal of the nail.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a division of U.S. application Ser. No.08/921,771, filed Aug. 15, 1997, pending, which is a continuation ofU.S. application Ser. No. 08/528,302, filed Sept. 14, 1995, abandoned.

BACKGROUND

1. Field of Invention

This invention relates to the medical treatment of diseased nails, clawsor hoofs (i.e., unguis), and specifically relates to the treatment ofdiseased nails by facilitating penetration of medication through thenail.

2. Statement of the Art

Humans and animals alike are commonly plagued by the infiltration ofmicro-organisms beneath the nail, claw or hoof which results in adisease condition causing pain, discoloration, and frequently loss ofthe unguis. In humans, for example, some diseases which attack the nailor nail bed can be treated fairly successfully with the use of cleansingand/or antiseptic preparations, while other diseases require treatmentby such means as systemic drug therapy. Nonetheless, many diseaseconditions of the nail, particularly onychomycosis (i.e., fungaldiseases), have had a relatively low success rate for treatment due tothe intransigent nature of the infectious micro-organisms.

It has been estimated that greater than twenty percent (20%) of thepopulation of the United States over the age of 40 suffers fromonychomycosis of the fingernails or toenails. The disease is known tooccur to a lesser extent in people below the age of forty, but theoccurrence of disease is still significant. Unfortunately, the currentmodalities for treatment of onychomycosis show a very low success rate.

Common means of treating microbial diseases, including onychomycosis,include oral administration of drugs and laser therapy. Laser therapy,as yet, is not well-developed nor widely practiced, and is veryexpensive because it must be conducted in a doctor's office by a trainedtechnician. Systemic drug therapy through oral administration has alsoproven to be relatively unsuccessful because of drug intolerances, theexpense of the medications and low patient compliance.

The most common means of treating onychomycosis is to remove the nailcompletely and topically apply medication to the underlying nail bed.However, not only is such treatment cosmetically unsightly, but thefungus which invades the nail often remains in the matrix of the fingeror toe (where the nail is formed) and the disease reoccurs immediatelyupon, or during, ingrowth of the new nail.

Undoubtedly, treatment of diseases involving nails would be greatlyenhanced by the ability to access the area around and below the nail, aswell as to penetrate the nail itself, without having to remove the nail.However, the thick and/or hardened nature of nails renders accessthrough, and to the area below, the nail very difficult. The same can besaid of diseases involving the claws or hooves of animals.

The usefulness of antifungal drugs in treating onychomycosis has beenlimited heretofore because of resistance to penetration of the nail orbecause of limited access to the nail bed through the nail. Thus, itwould be an improvement in the art to provide means for enhancingpenetration of the nail so that treatment of the nail with medicationmay occur at the situs and without having to remove or otherwisesignificantly damage the nail. It would also be advantageous to providesuch penetration means at a reasonable cost to the consumer, and in aform which would facilitate and encourage proper and consistent self-useby the afflicted person.

The art has disclosed the use of various substances as permeationfacilitators in drug delivery systems, but such permeation enhancershave only been used in connection with permeation of the epidermis.Examples of such methods and systems have been disclosed in U.S. Pat.No. 5,156,846; U.S. Pat. No. 5,296,222; French Patent Publication No.2,448,903; and French Patent Publication No. 2,556,218. The permeationof thickened nails, or other unguis, presents unique difficulties notencountered in permeation of the epidermis. To date, no means have beendeveloped to enhance permeation of nails for effective treatment ofdiseases involving nails, or other unguis.

SUMMARY OF THE INVENTION

In accordance with the present invention, nail-permeable medicationmeans are provided for facilitating the penetration of nails, claws,hoofs, or other hardened tissue derivatives of the skin or dermis, withappropriate medicaments to promote treatment of disease conditions whichoriginate or exist, in whole or in part, in or below the hardened tissueform. The nail-permeable medication means of the present invention isapplicable to many types and forms of hardened tissue, as describedabove, but use of the invention on the human nail is describedhereinafter as an exemplar.

The nail-permeable medication means of the present invention comprisesan effective amount of an enzymatic composition, in sufficientconcentration to modify the tissue structure of the nail to enhancepermeation of a medicament therethrough, and an effective amount ofmedicament. The enzymatic composition contains a proteolytic enzymeselected to be capable of temporarily modifying the cellular structureof the nail in a manner to permit permeation of medication through thenail without permanently harming (e.g., denaturing, degrading ordestroying) the structural integrity of the nail. The proteolytic enzymepreferably may be selected from the group comprising papain, bromelain,chymotrypsin, trypsin, or combinations thereof. However, otherproteolytic enzymes may be equally suitable. Other substances may beadded to the proteolytic enzyme in formulating the enzymaticcomposition, including activators, solubilizers, buffering agents,chelating agents, preservatives, thickening agents, colorants,permeation facilitators, etc. Water may or may not be a component of theenzymatic composition. Commercially available formulations containingproteolytic enzymes in an appropriate concentration, such as Sorenzymen™(Sorenson Pharmaceutical, Inc.) or Panafil® (Rystan Company), may besuitable for use in the invention.

The nail-permeable medication means also includes a medicament which isespecially selected for treatment of a particular disease condition. Theconcentration of medicament in the nail-permeable means is sufficient toeffectively control or eliminate the disease condition. Any medicamentmay be used in the invention provided that the medicament does not reactadversely with the proteolytic enzyme to render either the enzymaticcomponent or the medicament ineffective in facilitating permeation ofthe nail-permeable medication means through the nail. It is notable inthat respect that many medications have a water base, or have a watercomponent in addition to certain activators (e.g. thiols and mercaptanswhich include cysteine and a chelating agent, such as EDTA[ethylenediaminetetraacetic acid]) which, when admixed with theenzymatic composition, may begin to degrade the enzyme and/or reduce itseffectiveness. However, admixture of a water-based or water-containingmedicament with a proteolytic enzyme will typically degrade theenzymatic component slowly enough that significant penetration of thenail still takes place and the medicament is successfully delivered tothe disease site. Generally, papain and other proteolytic enzymes areeffective for approximately twenty-four hours after mixture with waterwhere certain activators are present. In the absence of such activators,papain may remain active for up to two years.

Examples of drugs which may be used in the nail-permeable medicationmeans include antibacterial, anti-viral, antifungal and otherantimicrobial compositions. Such drugs may also be ionic, anionic,nonionic, cationic, zwitterionic, or ampholytic. Suitable drugs for usein the nail-permeable medication means include ciclopirox olamine,miconazole, tolnaftate, terbinafme, amorolfm and econazole, as well asother drugs. It is possible to use pure forms of such drugs in formingthe nail-permeable medication means or commercial preparations of suchdrugs (e.g. in cream or lotion form) may be used.

The nail-permeable medication means is applied to a nail infected with adisease condition by bringing into contact with the nail the proteolyticenzyme composition and the selected medicament. The nail and surroundingarea is preferably first cleaned. Additionally, the nail may be filed orsanded and/or may be moistened for a short while before application,depending on the thickness or horniness of the nail, to enhancepenetration of the medicament therethrough. In most instances, the nailwill be moist anyway because of the wearing of socks or other footcoverings. The nail-permeable medication means is maintained in contactwith the affected nail for a period sufficient to permit penetration ofthe enzymatic component and medicament through the nail. The period ofcontact may typically be twenty-four hours. During that time the treatedarea may be preferably occluded.

The separate enzymatic component and medicament component may preferablybe admixed together at a ratio of from about 25:75 to 75:25, with apreferred ratio of about 50:50. The separate components may preferablybe admixed together immediately before application to the nail.Alternatively, the two components may preferably be pre-mixed, providedthat the addition of the medicament to the enzymatic component does notappreciably degrade the enzymatic component. The process of admixing thecomponents and applying them to the affected nail area may be repeatedas necessary and as permitted by the safety and efficacy indications ofthe drug and the enzymatic component.

The means of delivering the nail-permeable medication means to thediseased nail area may be accomplished in several manners. As acommercial tool to most conveniently package the components, and toaccurately measure for the user the correct amount of components, asystem may be used which comprises a first compartment containing theproteolytic enzyme component in a pre-measured amount, a secondcompartment containing a pre-measured amount of the appropriatemedicament component, and an intervention means between the twocompartments which enables a combination of the component from onecompartment and the contents of the other compartment for mixing. Thecompartment in which the mixing of the components takes place may alsohave an opening which permits the admixture to be squeezed from thecompartment onto the affected area. Occlusion means may also beassociated with the two-compartment system for occluding the nail areato which the permeable medication means has been applied.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an absorbance spectrum of a first human toe nail sample whichwas hydrated in heavy water and scanned over a twenty hour period;

FIG. 2 is an absorbance spectrum of a second human toe nail sample whichwas hydrated in heavy water and scanned over a twenty-two hour period;

FIG. 3 is a chart of ratios of C═O bond stretching and C--H bond bendingcalculated from scanning information derived from hydrated human toenails from the first human toe nail sample and from the second human toenail sample;

FIG. 4 is an absorbance spectrum of a human toe nail sample to which wasapplied varying amounts of a proteolytic enzyme composition;

FIG. 5 is an absorbance spectrum of a human toe nail sample to which wasapplied a four milligram amount of a proteolytic enzyme composition;

FIG. 6 is an absorbance spectrum of a human toe nail sample to which wasapplied a four molar (4M) solution of urea;

FIG. 7 is a representational illustration of a diffusion cell in whichhuman nails may be maintained in a simulated in vivo environment;

FIG. 8 is a plan view of the donor compartment illustrated in FIG. 7;

FIG. 9 is a standard calibration curve for ciclopirox olamine;

FIG. 10 is a standard ciclopirox olamine chromatographic peak;

FIG. 11 is a computer-identified and quantified sample peak;

FIG. 12 is an unidentified peak in the expected location for ciclopiroxolamine; and

FIG. 13 is a view in cross section of an exemplary device for mixing thedisclosed composition and occluding the wearer's skin after application.

DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION

The nail-permeable medication means is comprised of a proteolytic enzymecomponent and a medicament component. The proteolytic enzyme componentof the nail-permeable medication means acts to modify the nail tofacilitate penetration of the medicament through the nail. Theproteolytic enzyme component includes a proteolytic enzyme such aspapain, bromelain, chymotrypsin, or other suitable proteolytic enzymes,or combinations thereof. Papain may be a particularly suitable enzymefor inclusion in the nail-permeable medication means. The proteolyticenzyme component may also include substances which enhance theeffectiveness of the proteolytic enzyme, including enzyme activatorssuch as cysteine, and permeation facilitators such as urea. Theproteolytic enzyme component may also include various other materialswhich render the nail-permeable medication means effective forpenetrating the nail, including one or more alcohols. Water may beincluded in the proteolytic component.

The amount or concentration of enzyme in the proteolytic enzymecomponent is sufficient to facilitate penetration of medication into andthrough the nail, but is not so great that the nail tissue isirreparably damaged (i.e., will not rejuvenate or regenerate). In otherwords, it is well-known that proteolytic enzymes in higher concentrationcan cause various degrees of damage to skin tissue. In fact, proteolyticenzymes in higher concentrations are used to debride necrotic tissue inburn patients. However, the appropriate amount of proteolytic enzyme inthe nail-permeable medication means must be particularly selected topermit permeation through the hardened keratin tissue, the significantconstituent of nails, without resulting in irreparable damage to thenail.

Keratin is a protein formed by regularly repeated groupings of aminoacids which form long chains. The chains are held together by lateralbonds, including acid-base, hydrogen and van derWaals and disulfidebonds. The exact mode or action of proteolytic enzymes on the keratinmolecule of nails is not completely understood, but it appears thatproteolytic enzymes affect the bonds of the keratin molecules. Hydrationof the nail may also modify the keratin structure, such as by swelling.Absorbance spectrum studies were conducted on sample human toe nails tobetter understand the absorbance of papain and papain-based proteolyticenzyme compositions on such nails.

The toe nails were placed under vacuum (10⁻⁴ Torr) for five days todesiccate the samples, and were stored in sealed vials with a desiccantuntil used. The nails were then hydrated, using heavy water (D₂ O), forup to twenty-nine hours. Various spectra on the nails were obtainedthereafter as a function of time. FIG. 1 illustrates a spectrum obtainedon a first deuterated toe nail, and FIG. 2 illustrates a spectrumobtained on a second deuterated toe nail. The amide I band (center ofgravity) increased in intensity (absorbance) within two hours, and thenremained constant (FIGS. 1 and 2 ). The spectrum did not exhibit furtheralterations in bandwidth, presence or absence of deconvoluted underlyingbands or other spectral alterations as a function of time, confirmingthat hydration alone does not result in conformational alterations. Theamide I center of gravity (1631 cm⁻¹ [C═O stretching]) compared to theC--H bending (1453 cm⁻¹) increased within two hours and then remainedconstant, as shown in FIG. 3, indicating that hydration occurred inapproximately two hours.

Absorbance spectra were then obtained on hydrated toe nails to which wasapplied a papain or papain-based proteolytic enzyme composition for aperiod of three hours each. Toe nails were hydrated in heavy water forthree hours. Various spectra were then obtained. FIG. 4 illustrates thecomparative spectrum of a toe nail, beginning with a baseline spectrum20 of the hydrated nail. FIG. 4 also illustrates a spectrum 22 of thehydrated nail to which was applied one milligram of a proteolytic enzymecomposition containing six percent (6%) by weight papain (Marcor 30,000USP units/mg, or equivalent activity), ten percent (10%) by weight urea,fifty-two percent (52%) by weight water and six percent (6%) by weightpropylene glycol. A third spectrum 24 is plotted on the hydrated nail towhich was applied three milligrams of the same proteolytic enzymecomposition and a fourth spectrum 26 to which was applied an amount inexcess of three milligrams of the same proteolytic enzyme composition. Ashift in the amide I band suggested a redistribution or modification ofα-helical conformations of the keratin, perhaps at the expense ofβ-conformations. A shift in the amide I band with excess application ofthe proteolytic enzyme composition suggests a substantial increase inrandom β-conformations at the expense of a-helical conformations.

FIG. 5 illustrates another spectrum of a nail which was hydrated forthree hours with heavy water and was then contacted with a thin film offour milligrams of the same proteolytic enzyme composition previouslydescribed. Scanning took place over a twenty hour period with theresults that a band shift to lower wavelength values was observed withinone to two hours following application with an eventual shift back tothe original wavelength value at twenty hours. The affect of 4 molarurea (which is at substantially higher concentration than in the presentcomposition) on toe nails hydrated for three hours in heavy water, asillustrated in FIG. 6, resulted in a significant band shift towardhigher wave numbers. Both papain and urea appear to cause a furthermodification of the protein conformational populations than with ureaalone, or in bonding configurations, which allows movement of drugmolecules through the nail.

The proteolytic enzyme component of the nail-permeable medication meansmay contain from about 0.1 percent to about fifteen percent by weight ofan enzyme, with a preferred amount being from six percent to ten percentby weight. The amount of proteolytic enzyme that is effective in theproteolytic enzyme component is affected to some degree by the activityof the proteolytic enzyme which is used in the composition. With respectto papain, for example, the activity of the enzyme may vary from sourceto source, and may be measured by different units of activity dependingupon where the papain is purchased from or who the manufacturer is.

The United States Pharmacopeia (ASP) has developed one standard of unitmeasurement of papain activity where one (1) USP Unit of papain activityis the activity which releases the equivalent of 1 μg (microgram) oftyrosine from a specified casein substrate under the conditions of theassay, using the enzyme concentration that liberates 40 μg of tyrosineper ml (milliliter) of test solution. USP papain contains at least 6,000USP units per mg of papain. By contrast, for example, the RystanPharmaceutical Company, manufacturer of Panafil®, an enzyme-containingmaterial (papain), has established its own activity measurement ofpapain. One Rystan Unit is that quantity which under specifiedconditions will clot 10 microliters of milk substrate in one minute at40° C. Rystan Panafil®-White ointment is indicated as having 10,000Rystan Units of enzyme activity per milligram of ointment. The RystanPharmaceutical Company reports that its product has the equivalent of521.7 USP Units of activity.

Still other manufacturers, such as Sigma (Gaithersburg, Md.), indicatesthat its papain product contains approximately eighty percent (80%)protein and has an activity of 10-20 BAEE Units per milligram ofenzyme-containing material. One BAEE Unit is defined as the quantity ofpapain that will hydrolyze 1.0 micromoles of BAEE(N-4-benzoyl-L-argenine ethyl ester) per minute at 25° C. and pH 6.2.The ICN Company (Costa Mesa, Calif.) has three forms of papainavailable, one being 2× crystallized powder containing 10-20 BAEE Unitsof activity, the second being a 2× crystallized suspension formcontaining 15-40 BAEE Units and the third being a papain crude latexcontaining 1,750 USP Units/mg. No known means of correlating theseactivities is known to the inventors.

In formulating an exemplar proteolytic enzyme component, crude papainlatex (uncut), typically containing 40,000 to 60,000 USP Units ofactivity per milligram, was dissolved in water andprecipitated/crystallized by drop wise addition of ethanol, coupled withrefrigeration, to isolate crystalline papain from the latex.Approximately thirty-three percent (33%) of the crude latex wasconverted into a crystalline/amorphous mixture, and from that it wasestimated that approximately 20-25% yield of crystalline papain could berecovered from uncut papain latex upon 2× crystallization.

A particularly suitable formulation of the proteolytic enzyme componentof the nail-permeable medication means is the following:

EXAMPLE I

    ______________________________________                                        INGREDIENT          % BY WT.                                                  ______________________________________                                        Urea                10.0                                                        Papain.sup.1  6.0                                                             Glyceryl Sterate, PEG-150 Stearate 3.1-4.5                                    Cetyl Alcohol 2.4-3.5                                                         Stearic Acid 2.4-3.5                                                          Isopropyl Myristate 6.3-9.0                                                   Dimethicone 0.1-0.2                                                           Stearyl Alcohol 1.9-2.8                                                       Boric Acid 51.4-61.3                                                          Sodium Borate 0.8-1.0                                                         Propylene Glycol 4.2-6.0                                                      Triethanolamine 0.2-0.3                                                       Phenoxyethanol, methylparaben, 0.7-1.0                                        ethylparaben, propylparaben                                                   butylparaben (Phenonip) 1.5                                                 ______________________________________                                         .sup.1 Marcor Company (Hackensack, N.J.) papain having 30,000 USP Units/m     activity.                                                                

The foregoing formula for a proteolytic enzyme contains urea which hasbeen demonstrated to enhance permeation of the skin. In a proteolyticenzyme component formulation including urea, the concentration of ureamay be as high as 40% or greater, but is preferably in the range of 10%to 20%.

The medicament component of the nail-permeable medication means maycontain any type of drug or medicament which is necessary and effectiveat modifying, controlling or curing a disease condition of the nail andthe surrounding area. Such medicaments may include anti-bacterialcompositions, anti-mycotic or antifungal compositions, anti-viralcompositions, or any other suitable medicament. Exemplar medicamentswhich are useful in the nail-permeable medication means are ciclopiroxolamine, miconazole, itraconazole, clotrimazole, bifonazole,terbinafine, amorolfin, griseofulvin, econazole, tolnaftate and mixturesthereof. Effective amounts of these medicaments in the nail-permeablemedication means will vary depending upon the disease condition to betreated. However, for most disease conditions, particularly fungaldiseases, the minimum amount of medicament which will inhibit the growthor activity of a fungus (e.g., Trichophyton rubrum or Trichophytonmentagrophytes) is in the range of about 0.007 micrograms per milliliterto about 10 micrograms per milliliter. Specifically, for example, theminimum inhibitory concentration (MIC) of ciclopirox olamine withrespect to T. rubrum is 10 μg/ml.

The efficacy of the nail-permeable medication means of the presentinvention was first evaluated by conducting comparative studiescomprising application of a selected drug applied to a human nail invitro as compared to the application of the nail-permeable medicationmeans containing the same drug applied to a human nail in vitro.Specifically, these tests were conducted using diseased human toenailswhich were surgically removed from patients as a means of curing thedisease condition of the nail.

Each nail was maintained in an environment simulating an in vivocondition. Each nail was maintained suspended on a plastic holder whichallowed air to circulate about the nail. Each nail holder was retainedin a separate enclosed system the temperature and humidity within whichcould be carefully regulated to simulate the human body. Theseartificial in vivo environments provided the diffusion cells in whichthe studies were conducted.

Human nails were prepared by cleaning and, in some instances, grindingthe nail down to establish a more even thickness across the nail. Thenails were then hydrated using distilled water to render the nailspliable for placement in the diffusion cell. The prepared and hydratednails were placed into nail diffusion cells 10, as illustrated in FIG.7, by positioning the nail 12 over the nail saddle 14. A Teflon® donorcompartment 16 was positioned over the nail 12 and the nail saddle 14was joined to the donor compartment 16 by placement of a siliconesealant about the edges of the nail 12 and donor compartment 16. Afterthe silicone cured, water leakage was tested and the nails were resealedif necessary. Care was taken in the application of the sealant to ensurethat this material did not ooze into the nail absorption area as it wasbeing clamped into place. Attempts were made to use an appropriate size"O" ring 18 to form the seal between the nail saddle 14 and the donorcompartment 16.

The cell 10 was used by adding solvent, usually de-ionized water, untilthe solvent came into contact with the nail. Fluid pre-treatment ortreatment products were dropped into the port 22 of the donorcompartment 16 as shown in FIG. 8, which illustrates a top view of thedonor compartment 16. When semi-solid treatment formulations were used,excess product was removed from the nail by cleansing with cotton swabsafter twenty-four hours and prior to application of another nailtreatment or pre-treatment product. Pretreatment products (e.g.,hydrating fluid, etc.) and treatment products (i.e., nail-permeablemedication means) were changed or supplemented daily throughout theexperimental period.

After addition of the pre-treatment or treatment product, the opening ofthe donor cell 10 was occluded with silicone cement/sealant. Thepossibility of entrapped air at the interface of the receiver solventand nail was checked daily, as well as at the beginning of theexperiment. To control this problem, care was taken to be certain thatno bubbles were present at the time solvents were added and the nailplaced in the saddle. Bubbles may not be visible after the apparatus isfully assembled. Also, dissolved gases tend to separate into bubbles andrise to the nail/receiver interface during the process. It was foundthat tipping the apparatus into a horizontal position and tapping thecell gently did not interfere with the stirring or diffusion process andallowed the bubbles to escape through the sampling port 24.

Ciclopirox olamine (6-Cyclohexyl 1-hydroxy 4-methyl 2(1H)-pyridone,2-aminoethanol salt [Bertrafen®]), an antimycotic drug, was used in thenail-permeable medication means of the invention for the purposes oftesting and analysis. Three analytical methods are identified in theliterature which appear to be acceptable means of determining drugdiffusion rates. One method requires the use of radio-labeled drug, andanother method utilizes LC (liquid chromatography) equipment. The thirdmethod, described by Beffiardo, et al., in "Micro-liquid chromatographymethod for the determination of ciclopirox olamine after pre-columnderivatization in topical formulations" Jr. Chromatography, Vol. 553(1991), p. 41-5, was the method used here. However, because of lack ofaccess to a micro-LC system, lesser concentrations of ciclopirox olamine(<1-2 μ/ml of diffusion fluid) were unquantifiable. Lesserconcentrations were sometimes seen as small irregularities on the HPLC(high-pressure liquid chromatography) tracing, but the identity was notconfirmed by computer. In each analysis, from 1-3 ml of thenail-permeable medication means, as described further below, insubstantially fluid form, was lyophilized and used for derivatizationand analyses. The volume lyophilized depended on anticipatedconcentrations of ciclopirox olamine. Other quantities and volumes asreported in the publication were the same as used by the authors of theBelliardo article.

Computer-generated calibration curves were obtained prior to each runusing a standard solution prepared as described by Belliardo. A standardcalibration curve is shown in FIG. 9 and a standard ciclopirox olaminepeak is shown in FIG. 10. A computer-identified and quantified samplepeak is shown in FIG. 11, and an unidentified peak in the expectedlocation is shown in FIG. 12.

The nail-permeable medication means of the invention was prepared byformulating a proteolytic enzymatic component and a medicamentcomponent. The proteolytic enzyme component was prepared by making acomposition containing a proteolytic enzyme and other additives. Papainwas used in the proteolytic enzyme component of the nail-permeablemedication means in all of the experiments. However, other proteolyticenzymes, such as bromelain and chymotrypsin are suitable for use in theinvention. In most experiments, the concentration of papain was 0.5%Sigma papain (Sigma Co., product no. P4762 [2× crystallied, lyophilizedpowder, 16-20 BAEE units/mg]). Several experiments were conducted inwhich other concentrations (0.1%, 0.25% and 1.0%) of papain were used.In most studies, the 0.5% concentration papain produced optimaldiffusion results.

Papain solutions also contained the activators l-cysteine (Sigma Co.,product C-7755 [crystalline]) in a concentration of 0.28% and EDTA(Sigma Co. product EDS) in a concentration of 0.24%. All solutions wereprepared fresh daily due to the instability of papain when dissolved inthe activators. Unused portions of solutions were discarded if not usedwithin one or two hours after preparation. Solutions were prepared bysimple solution in water or mixtures of water and ethanol, althoughother alcohols may be substituted, including isopropyl. Most experimentswere conducted using water alone as the solvent without buffering. In afew experiments, ethanol was mixed with water in 25:75 and 50:50 ratios.Some experiments also used buffered solutions. Buffering wasaccomplished with known phosphate buffers adjusted to pH 4.0, 7.4 and10.0. In some later experiments a commercial form of papain (Panatil®Ointment, Rystan Co.) was used where papain concentrations wereexpressed in terms of USP units. The commercially available form ofpain, Panafil®, contains 521 USP units of papain per mg of product.

Most experiments were conducted using 1.0% weight per volume ofciclopirox olamine (Sigma Company) dissolved in water or inethanol/water mixtures in a ratio of 25:75 or 50:50. In someexperiments, commercial forms of ciclopirox olamine were used. Theseincluded Loprox® from Hoechst-Roussel Company, in both lotion and creamforms. The enzyme component and drug component were combined at a ratioof about 50:50.

Some of the experiments were conducted by saturating felt pads with apapain solution including activators, as described above. The pads wereimmediately lyophilized (freeze dried) after soaking. In some of theexperiments, a solution of papain without activators was also preparedand used to saturate pads which were lyophilized immediately. Whenlyophilized pads were used in the experiments, they were hydratedimmediately preceding application of the pad to the nail.

SUMMARY OF IN VITRO STUDIES

The initial diffusion studies evaluating the nail-permeable medicationmeans of the invention used papain (0.5% Sigma papain, product no.P4762) as the proteolytic enzyme and ciclopirox olamine as themedicament component. The diffusion studies were carried out by soakingthe human nails in a 0.5% papain-plus-activators solution for 24 hoursbefore placing them in the nail diffusion cells and measuring drugdiffusion. As soaking time was increased from one day to three days, theamount of drug that diffused through the treated nails also increased.The papain-plus-activators, as well as the ciclopirox olamine drug, weredissolved in distilled water. Hydroalcoholic solutions as a solvent forthe drug were also tried. The overall effect of these changes was thatincreasing soaking time of the proteolytic enzyme component containingpapain-plus-activators, and the inclusion of an alcohol, such as ethanolin a concentration of at least twenty-five percent, in the solvent forthe drug increased the amount of drug that diffused through thenailplate (ranges of 2-6 μg/ml increased to 3-9 μg/ml in a seven-daydiffusion period). Control studies in which the nails were not contactedwith any proteolytic enzyme component showed no diffusion of drugthrough the nail after seven (7) days of treatment, as determined byHPLC analyses. Solvents used for these studies included water, 25%ethanol in water and 50% ethanol in water.

Because it is impossible to soak toenails in clinical use, theproteolytic enzyme component containing papain-plus-activators was addedto the diffusion cells on top of the nails as a solution for three days,after which the diffusion of the drug was measured for seven days(sample nos. 53-90). The amount of drug diffused was less than when thenails were soaked with the papain-plus-activators. It was also observedthat the presence of alcohol in the solution for the drug increased drugdiffusion for the seven-day period (ranges of 2-6 μg/ml). Increasing theconcentration of the papain did not increase the amount of drugdiffused.

In another group of tests (sample nos. 91-118), felt pads were used forpapain as well as drug application to the nails. In some cases the padswere soaked with the solutions and used in that form. In other tests(sample nos. 119-154), the pads were saturated with the solution andthen lyophilized before use. The use of saturated pads as a three-daypretreatment and seven-day drug diffusion gave the best results (0.5-4μg/ml). Several combinations of differing papain concentrations as wellas methods and applications were tried using lyophilized pads. Theinclusion of papain, activators and drug all in the same pad was tried(sample nos. 168, 169). The most successful method was that in which0.5% Sigma papain and 1% drug in a lyophilized pad was hydrated with asolution containing activators after the pad was placed on the nail.Concentrations of diff-used drug over a 21-day period were 1-3 μg/ml.

The results of the various tests conducted with the proteolytic enzymecomponent and the medicament component are shown in Table 1, as follows:

                                      TABLE 1                                     __________________________________________________________________________    SAMPLE                                                                             PAPAIN               CICLOPIROX           ANALYSES                       NO.  CONC. %                                                                            SOLVENT                                                                             MODE                                                                              DURATION                                                                            CONC. %                                                                            SOLVENT                                                                             MODE                                                                              DURATION                                                                            (ug/ml)                                                                             COMMENTS                 __________________________________________________________________________     53  0.5  W     S   1D    1    W     T   7D    7                                 54 0.5 W S 1D 1 W T 7D 2                                                      55 0.5 W S 1D 1 W T 7D 0                                                      56 0.5 W S 1D 1 50A T 7D <.3                                                  57 0.5 W S 1D 1 50A T 7D <.3                                                  58 0.5 W S 1D 1 50A T 7D 0                                                    62 0.5 W S 1D 1 25A T 7D 2                                                    63 0.5 W S 1D 1 25A T 7D 4                                                    64 0.5 W S 1D 1 50A T 7D 2                                                    65 0.5 W S 1D 1 50A T 7D 2                                                    66 0.5 W S 1D 1 50A T 7D 1                                                    67 0.5 W S 1D 1 50A T 7D 0                                                    68 0.5 W S 1D 1 25A T 7D 0                                                    69 0.5 W S 1D 1 25A T 7D 5                                                    70 0.5 W S 1D 1 25A T 7D 2                                                    71 0.5 W S 1D 1 25A T 7D 0                                                    72 0.5 W S 1D 1 25A T 7D <.3                                                  73 0.5 W S 2D 1 W T 7D 4                                                      74 0.5 W S 2D 1 W T 7D 2                                                      75 0.5 W S 2D 1 W T 7D 0                                                      76 0.5 W S 2D 1 25A T 7D 6                                                    77 0.5 W S 2 1 25A T 7D 3                                                     78 0.5 W S 2D 1 25A T 7D <.3                                                  79 0.5 W S 2D 1 50A T 7D <.3                                                  80 0.5 W S 2D 1 50A T 7D <.3                                                  81 0.5 W S 2D 1 50A T 7D 4                                                    82 1 W S 1D 1 W T 7D 7                                                        83 1 W S 1D 1 W T 7D 3                                                        84 1 W S 1D 1 W T 7D <.3                                                      85 0.5 W S 1D 1 50A T 7D 4                                                    86 0.5 W S 1D 1 50A T 7D 3                                                    87 0.5 W S 1D 1 50A T 7D 3                                                    88 2 W S 1D 1 50A T 7D <.3                                                    89 2 W S 1D 1 50A T 7D <.3                                                    90 2 W S 1D 1 50A T 7D <.3                                                    91 0.5 W FP* 2D 1 W FP* 7D <.3                                                92 1 W FP 2D 1 W FP 7D 0                                                      93 1 W FP 2D 1 W FP 7D 0                                                      94 0.1 W FP 1D 1 50A T 7D 0                                                   95 0.1 W FP 1D 1 50A T 7D 0                                                   96 0.25 W FP 1D 1 50A T 7D <.3                                                97 0.25 W FP 1D 1 50A T 7D <.3                                                98 1 W FP 2D 1 50A FP 7D <.3                                                  99 1 W FP 2D 1 50A FP 7D <.3                                                 100 1 W FP 2D 1 50A FP 7D <.3                                                 101 0.5 W FP 3D 1 25A FP 7D 0                                                 102 0.5 W FP 3D 1 25A FP 7D 1                                                 103 0.5 W FP 3D 1 25A FP 7D 2                                                 104 0.5 W FP 3D 1 50A FP 7D 0                                                 105 0.5 W FP 3D 1 5OA FP 7D 4                                                 106 2 W FP 3D 1 50A FP 7D 4                                                   107 2 W FP 6D 1 W FP 7D 1                                                     108 2 W FP 6D 1 W FP 7D 0                                                     109 0.5 W FP 6D 1 W FP 7D 2                                                   110 0.5 W FP 3D 1 W FP 7D 1                                                   111 0.5 W FP 3D 1 W FP 7D 34**                                                112 0.5 W FP 3D 1 25A FP 7D 1                                                 113 0.5 W FP 3D 1 25A FP 7D 3                                                 114 0.5 W FP 3D 1 50A FP 7D 3                                                 115 0.5 W FP 3D 1 50A FP 7D 2                                                 116 0.5 W FP 3D 1 50A FP 7D 0                                                 117 0.5 W FP 3D 1 50A FP 7D 0                                                 118 0.5 W FP 3D 1 50A FP 7D 0                                                 119 0.5 W LFP 3D 1 W LFP 7D 0***                                              120 0.5 W LFP 3D 1 W LFP 7D 0***                                              121 0.5 W LFP 3D 1 W LFP 7D 0***                                              122 0.5 W LFP 3D 1 W LFP 7D 0***                                              123 0.5 W LFP 3D 1 W LFP 7D 0***                                              124 0.5 W LFP 3D 1 W LFP 7D 0***                                              128 0.5 W LFP 3D 1 W LFP 7D 0****                                             129 0.5 W LFP 3D 1 W LFP 7D 0****                                             130 0.5 W LFP 3D 1 W LFP 7D 0****                                             131 0.5 W LFP 3D 1 W FP 7D 0                                                  132 0.5 W LFP 3D 1 W FP 7D 0                                                  133 0.5 W LFP 3D 1 W FP 7D 0                                                  140 0.1 W LFP 3D 1 W FP 7D 1 Activators added in                                        hydration fluid                                                     141 0.1 W LFP 3D 1 W FP 7D 1 Activators added in                                        hydration fluid                                                     142 0.1 W LFP 3D 1 W FP 7D <.3 Activators added in                                      hydration fluid                                                     143 0.1 PG LFP 3D 1 W FP 7D 0 Propylene glycol as                                       hydration fluid (pap)                                               144 0.1 PG LFP 3D 1 W FP 7D 0 Propylene glycol as                                       hydration fluid (pap)                                               145 0.1 PG LFP 3D 1 W FP 7D 0 Propylene glycol as                                       hydration fluid (pap)                                               146 0.1 PG LFP 3D 1 W LFP 7D 0 Propylene glycol as                                      hydration fluid (pap)                                                         Not occl.                                                           147 0.1 PG LFP 3D 1 W LFP 7D 0 Propylene glycol as                                      hydration fluid (pap)                                               148 0.1 PG LFP 3D 1 W LFP 7D 0 Propylene glycol as                                      hydration fluid (pap)                                               149 0.1 W LFP 3D 1 50A LFP 7D 11.8 50A as hydration fluid                               for ciclopirox                                                      150 0.1 W LFP 3D 1 50A LFP 7D 3 5OA as hydration fluid                                  for ciclopirox                                                      151 0.1 W LFP 3D 1 50A LFP 7D 0 50A as hydration fluid                                  for ciclopirox                                                      152 0.5 W LFP 3D 1 50A FP 30D 2456 50A as hydration fluid                               for ciclopirox -                                                              Probable leakage                                                    153 0.5 W LFP 3D 1 50A FP 30D 0.5 50A as hydration fluid                                for ciclopirox                                                      154 0.5 W LFP 3D 1 50A FP 30D <.3 50A as hydration fluid                                for ciclopirox                                                      155 0.5 W FP 3D 1 W FP 30D 0.5 Activators added in                                      hydration fluid                                                     156 0.5 W FP 3D 1 W FP 30D <.3 Activators added in                                      hydration fluid                                                     157 0.5 W FP 3D 1 W FP 30D <.3 Activators added in                                      hydration fluid                                                     158 0.1 W FP 3D 1L L T 20D 0 Activators added in                                        hydration fluid                                                     159 0.1 W FP 3D 1L L T 20D 0 Activators added in                                        hydration fluid                                                     160 0.1 W FP 3D 1L L T 20D 0 Activators added in                                        hydration fluid                                                     161 0.1 PG FP 3D 1L L T 20D 0 Activators added in                                       hydration fluid                                                     162 0.1 W FP 3D 1L L T 20D 21.8 Activators added in                                     hydration fluid -                                                             Probable leakage.                                                   163 0.1 W FP 3D 1L L T 20D 0                                                  170 P P T 3D 1L L T 7D 0                                                      171 P P T 3D 1L L T 7D 0                                                      172 P + W P + W T 3D 1L L T 7D 0                                              173 P + W P + W T 3D 1L L T 7D 0                                              174 P + A P + A T 3D 1L L T 7D 0                                              175 P + A P + A T 3D 1L L T 7D 0                                              164 P P FP 3D 1L L T 40D 0                                                    165 P P FP 3D 1L L T 40D 0                                                    166 P P FP 3D 1L L T 40D 0                                                    167 P P T 6D 1L L T 40d 0 Activators added in                                           hydration fluid                                                     168 P + L P + L T 46D P + L P + L T 46D <.3 Panafil and Loprox                          used together on nails                                              169 P + L P + L T 46D P + L P + L T 46D <.3 Panafil and Loprox                          used together on nails                                              176 0.5 pH10 S 5D 1 W T 10D 0                                                 177 0.5 pH10 S 5D 1 W T 10D 0                                                 178 0.5 pH10 S 5D 6 W T 10D 250 Probably a leaker.                            179 0.5 pH10 S 5D 6 W T 10D 30 Probably a leaker.                             180 0.5 pH4 S 5D 1 W T 10D 0                                                  181 0.5 pH4 S 5D 1 W T 10D 75 Probably a leaker.                              182 0.5 pH4 S 5D 6 W T 10D 10                                                 183 0.5 pH4 S 5D 6 W T 10D 0                                                __________________________________________________________________________     *In experiments using felt pads, the pads were changed on a daily basis.      **Result may be due to leakage.                                               ***No activators in papain solution.                                          ****Activators added in hydration fluid (pap).                                KEY                                                                           W = Water                                                                     S = Soak                                                                      D = Days                                                                      L = Loprox Cream, 1.0%                                                        T = Topical application on nail                                               FP = Felt Pad                                                                 PG = Propylene Glycol                                                         50A = 50% Alcohol in water                                                    25A = 25% Alcohol in water                                                    LFP = Lyophilized felt pads                                                   P + W = Panafil and water mixed, 50% each                                     P + A = Activators (EDTA & Cysteine) added to Panafil Ointment                <.3 = Concentration less than 0.3 μg/ml, etc. Small blip, below HPLC       ability to identify and quantify.                                        

RESULTS OF CLINICAL STUDIES

A pilot clinical study was conducted by investigators in Salt Lake City,Utah and Miami, Florida consisting of approximately fifty patients whohad onychomycosis. Patients enrolled in the study were diagnosed withsubungual onychomycosis from a positive culture of a target nail. Thetarget nail was that nail most severely involved with symptoms ofonychomycosis. While the majority of patients included had previouslyreceived topical therapies for their onychomycosis, patients treatedwith other topical anifungals within 14 days, or systemic treatmentswithin six months prior to study entry, were excluded from the study.Patients with a history of non-response to standard antimycotictreatments, or severe abnormalities or deformities of the assessedfingers or toes, were excluded. Also, patients who had demonstratedprevious intolerance to either the antifungal agent or other proteolyticenzymes, who had vascular compromise of their extremities, or hadintrinsic nail disease were excluded.

Patients chosen for the study were randomly placed into one of the fourstudy arms shown in Table 2. The protocol called for an eleven monthtreatment period.

                  TABLE 2                                                         ______________________________________                                        ARM         APPLICATION                                                       ______________________________________                                        1           Proteolytic enzyme.sup.1 pretreatment for 2                          weeks followed by                                                             antifungal.sup.2 treatment B.I.D.                                            2 Concurrent, B.I.D. treatment with                                            nail-permeable medication means.sup.3                                        3 B.I.D. treatment with antifungal.sup.2 alone                                4 Concurrent Q.D. treatment with                                               nail-permeable medication means.sup.3                                      ______________________________________                                         .sup.1 The enzyme used for the studies was the formula described in           EXAMPLE I, above.                                                             .sup.2 The antifungal medicament used in the studies was ciclopirox           olamine in a concentration of 1%.                                             .sup.3 The nailpermeable medication means used in this study was a            preparation containing the proteolytic enzyme component described in          EXAMPLE I in admixture with ciclopirox olamine in a concentration of abou     10 mg/ml in a ratio of about 50:50.                                      

Patients visited their physicians on a monthly basis for evaluation.Clinical measurements and mycological cultures were taken on alternatingvisits. Adverse events were defined as any abnormal inflammation of thetissues surrounding the nail from the patient's pretreatment conditionand were checked by their physician or reported as they occurred.Patient evaluations of clinical treatment were obtained by a survey ofpatients. Ease of application, convenience of application, and patient'sassessments of the clinical results were rated by patients on a scale of1 to 5 with 1 indicating visible effect and 5 being positive. Inaddition, patients were also asked to respond to treatment compliance byreporting the number of days per week which they applied theirmedication as prescribed.

Due to previously reported high relapse rates of onychomycosis amongother investigators' studies, a follow-up study was conducted withpatients from this pilot clinical study who achieved clinical cure.Patients with clinical cure (target nail 100% clear of fungus followingtreatment) were randomly placed into one of two follow-up arms of thestudy as follows:

1. One-third of the patients received no treatment for follow-up, yetcontinued to be observed for reoccurrence of fungus for a period of sixmonths.

2. Two-thirds of the patients were asked to apply the medicationaccording to the arm 4 treatment regime (single daily concurrenttreatment using the permeation medicament) for one week per month, for aperiod of six months.

In addition to this follow-up, all failures from study arms 1 and 3 weremoved into arm 4 where they were treated with a dose of thenail-permeable medication means on a once-per-day basis. Arm 2 (Table 3)patients, upon complete cure, were also entered into the follow-up studyas outlined above.

Treatment of onychomycosis with topical antifungal agents has typicallynot resulted in either mycologic or clinical cure in the past.Therefore, patients treated in arm 3 (antifungal only) of the study wereused as the baseline upon which clinical and mycologic results weremeasured.

Despite good compliance by patients whose toes were treated in arm 1 ofthe study (Table 3), an average growth rate of clear nail ofapproximately 1 mm was observed over the first four months of the trial.The lack of clear nail growth over this period of time may be due to thelack of treatment with the nail-permeable medication means following theinitial two weeks of proteolytic enzyme component-only therapy. It wasalso observed that during the 2 week treatment of twice per dayapplication of proteolytic enzyme component, adverse reactions occurredin approximately 73% of the patients.

Compliance was also good among patients whose toes were treated in arm2, and the average growth was approximately 3 mm over the first fourmonths of the trial. These results showed that the proteolytic enzymecomponent had a positive affect in assisting penetration. The mycologiccure rate in this group was approximately 71%. Unfortunately, theincidence of adverse reactions in this group was approximately 67%. Thiswas presumably due to the twice daily application of the proteolyticenzyme component.

Patients treated in arm 3 (with antifungal only) felt that it was veryconvenient, yet they experienced no clear nail growth and either droppedout of the study due to a lack of result after five months or wereswitched to arm 4 of the study. This supports previous studies thatsuggest topical antifungal agents alone have minimal effect on eitherclinical or mycologic cure of onychomycosis. No adverse reactions werereported or observed in this patient population.

Patients in arm 4 showed excellent compliance, and also showed averageclear growth rates of approximately 3 mm at month 4, 4.4 mm at month 5,and 6 mm at month 7. Patients treated in arm 4 received treatment tofingers. It should be noted that finger nails grow at a more rapid pacethan do toe nails. Patients in arm 4 also showed a mycological cure rateof approximately 75% at month 7 and continued to show increasedimprovement as treatment continued. No adverse reactions were reportedor observed in this patient population.

                                      TABLE 3                                     __________________________________________________________________________       Clear %    Average Days                                                                         Ease                                                                              Convenience                                            Study Growth Adverse of Treatment of Use of Use Results % Cult.                                                 Arm (4 mos.) Reactions Per Week                                              -5) (1-5) (1-5) Neg.                       __________________________________________________________________________    1  1 mm  73   5.71   3.88                                                                              3.38  4.25                                                                              --                                           2 3 mm 67 6.80 3.8 3.8 4.2 71                                                 3 0 mm  0 6.50 5 5 1 --                                                       4 3.14 mm    0 6.75 4.63 4.75 4.2 75                                        __________________________________________________________________________

Preliminary clinical data and patient survey results are summarized inTable 3. These results suggest the following: First, the high incidenceof adverse reactions in arms 1 and 2 of the study, as illustrated inTable 3 indicate that twice daily applications of the proteolytic enzymecomponent alone are not acceptable. Although none of the adversereactions were severe, the high rate does interfere with patientcompliance and interrupts the daily regime.

Second, all arms using the nail-permeable medication means (1, 2, and 4)show both clinical and mycological improvement. Because of the intensiveapplication of the proteolytic enzyme component in the first two weeksin arm 1, there is an initial clear nail growth. However, patients inthis arm slow, or regress, over time showing only an average 1 mm ofclear nail growth at four months, as illustrated in Table 5. This islikely due to the terminated application of proteolytic enzyme componentafter two weeks. Clear growth in arms 2 and 4 continued to increase overtime. Patients also reported high satisfaction with results in all armsusing the proteolytic enzyme component.

Third, patients in all arms reported that the nail-permeable medicationmeans was both easy to use and a convenient form of application.Patients involved in the once a day application in arm 4 reported highersatisfaction in the survey. In all arms patients reported very highcompliance as summarized in Table 3. The overall average daily 5treatment rate was 6.4 days per week. Arm 4 reported application 6.75days per week.

The results of the pilot study indicated that concurrent, once a day,treatment with the nail-permeable medication means (arm 4) appears to beboth the safest and most effective regime for the treatment ofonychomycosis. Although the patients in the clinical studies applied apreformed nail-permeable medication means comprising a proteolyticenzyme component and a medicament component, it has been observed thatcomparable success rates for treatment of onychomycosis has resultedwhere patients have combined equal portions of the proteolytic enzymecomponent and the medicament component immediately prior to applicationto the diseased nail. Further, comparable success rates have beendemonstrated in patients who mixed equal portions of the proteolyticenzyme component and the medicament component directly on the nailsurface and surrounding area. In all manners of application, patientsapplied occlusion means to the treated nail area following applicationof the nail-permeable medication means or proteolytic enzyme componentand medicament component.

What is claimed is:
 1. A method for treating diseased animal nailscomprising:providing a preformed topically applied nail-permeablecomposition comprising a proteolytic enzyme component in admixture witha nail disease-affecting medicament component; topically applying saidnail-permeable composition to an area of diseased animal nail; occludingsaid area of diseased nail with occlusion means following application ofsaid composition; contacting said area of diseased animal nail with saidnail-permeable composition for a period of time sufficient to effectpermeation of said nail-permeable composition through said infectednail.
 2. The method of claim 1 wherein said composition is applied tosaid area of diseased animal nail once in a twenty-four hour period. 3.The method of claim 2 wherein said composition is contacted with saiddiseased animal nail for at least twenty-four hours.
 4. The method ofclaim 2 wherein said composition is contacted with said diseased animalnail for up to twenty-four hours.
 5. The method of claim 1 furthercomprising;removing said occlusion means; providing a subsequent dosageof said preformed nail-permeable composition; applying said subsequentdosage of said preformed nail-permeable composition to said area ofdiseased animal nail; and occluding said area of diseased animal nailwith occlusion means.
 6. A method for treating a disease condition of ananimal nail comprising:forming a nail-permeable composition by admixingtogether a proteolytic enzyme component composition and a naildisease-affecting medicament; immediately applying said formednail-permeable composition to an area of diseased-affected animal nail;occluding said area of diseased-affected animal nail with occlusionmeans following application of said composition; and contacting saidarea of disease-affected animal nail with said formed nail-permeablecomposition for a period of time sufficient to effect permeation of saidformed nail-permeable composition into said animal nail.
 7. The methodaccording to claim 6 wherein said formed nail-permeable composition iscontacted with the disease-affected nail for up to twenty-four hours. 8.The method according to claim 6 further comprising:removing saidocclusion means; forming a subsequent dosage of nail-permeablecomposition by admixing together a proteolytic enzyme composition and anail disease-affecting medicament; applying said subsequent dosage ofnail-permeable composition to said area of disease-affected animal nail;and occluding said area of disease-affected animal nail with occlusionmeans.
 9. A method for treating a disease condition in an animal nailcomprising:providing a topically-applied proteolytic enzyme compositioncontaining an amount of proteolytic enzyme sufficient to modify ananimal nail to facilitate permeation of a medicament through said animalnail without irreversibly damaging said nail; providing atopically-applied medicament component containing a sufficient amount ofnail disease-affecting medicament to affect a disease condition of saidanimal nail; applying an effective amount of said proteolytic enzymecomposition to said animal nail; simultaneously applying with saidproteolytic enzyme composition said medicament component to said animalnail; mixing said applied proteolytic enzyme component and said appliedmedicament component on said animal nail; and occluding said animal nailwith occlusion means.
 10. The method according to claim 9 wherein saidmixed proteolytic enzyme composition and mixed medicament component arecontacted with said animal nail for at least twenty-four hours.
 11. Amethod for treating a disease condition in animal nailscomprising:providing a topically-applied proteolytic enzyme compositioncontaining an amount of proteolytic enzyme sufficient to modify ananimal nail to facilitate permeation of a medicament through said animalnail without irreversibly damaging said nail; providing atopically-applied medicament component containing a sufficient amount ofnail disease-affecting medicament to affect a disease condition of saidanimal nail; applying an effective amount of said proteolytic enzymecomposition to said animal nail; contacting said proteolytic enzymecomposition with said animal nail for a period of time sufficient tomodify said animal nail to facilitate permeation therethrough; applyingsaid medicament component to said animal nail; and occluding said animalnail with occlusion means.
 12. The method according to claim 11 whereinsaid proteolytic enzyme composition is applied to said animal nail for aperiod of at least two hours prior to application of said medicamentcomponent.
 13. The method according to claim 12 wherein said proteolyticenzyme composition and said medicament component are contacted with saidanimal nail for a period of up to twenty-four hours.
 14. The methodaccording to claim 12 wherein said proteolytic enzyme composition issubstantially removed from said-animal nail prior to application of saidmedicament component.